Pyridin-2yl sulfanyl acid esters and process for the preparation thereof

ABSTRACT

The present invention relates to Pyridin-2-yl sulfanyl acid ester compounds having antiinflammatory properties. The present invention particularly relates to novel anti-inflammatory heterocyclic acid esters of Pyridin-2-yl sulfanyl having the structure of general formula 1 which have been screened for their antiinflammatory activity with respect to inhibition of adhesion of neutrophils, isolated from human peripheral blood, onto the surface of human umbilical vein endothelial cells (HU-VEC) as a result of inhibition of the cytokine-stimulated expression of cell adhesion molecule ICAM-1. The compound RS—Z, 3-(Pyridin-2-yl sulfanyl)-propionic acid pentyl ester (structure 1a, R 1 =H, R 2 =H, R 3 =CH2-COOC5H 11 ) was found to be most effective for ICAM-1 and neutrophil adhesion inhibition and was found to effectively alleviate inflammation mediated by excessive leukocyte infiltration leading to inflammatory disorders or like conditions, such as acute lung injury and acute respiratory distress syndrome in mice.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a national phase of International PatentApplication No. PCT/IN2011/000477, filed Jul. 20, 2011, which claimspriority to Indian Patent Application No. 1691/DEL/2010, filed Jul. 20,2010, the entire disclosures of which are incorporated by referenceherein.

FIELD OF THE INVENTION

Present invention relates to Pyridin-2-yl sulfanyl acid ester compoundsof general formula 1

whereinR₁=H;R₂=H, COOMe;R₃=(CH2)_(n)COOR₄;R₄=C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₁;n=1-7.

The present invention further relates to the synthesis of acid ester ofPyridin-2-yl sulfanyl compounds of general formula 1 (heterocyclic addesters of pyridine-2-yl sulphanyl or heterocyclic acid esters of2-pyridinylthio compounds).

Present invention further relates to compounds of general formula 1exhibiting potent anti-inflammatory activity with respect to inhibitionof adhesion of neutrophils isolated from human peripheral blood onto thesurface of human umbilical vein endothelial cells (HUVECs) as a resultof inhibition of the cytokine-stimulated expression of cell adhesionmolecule ICAM-1 (Intercellular cell adhesion molecule-1).

Present invention further relates to the synthesis and use of the mostactive compound (1a) RS—Z, 3-(Pyridin-2-yl sulfanyl)-propionic acidpentyl ester in mice to alleviate inflammation mediated by excessiveleukocyte infiltration leading to inflammatory condition or disorderssuch as acute lung injury, acute respiratory distress syndrome, septicshock, ischemia-hyperfusion etc.

BACKGROUND OF THE INVENTION

An analysis of the molecular structure of active anti-inflammatoryagents available in literature suggests that these compounds contain asub structure of pyridyl group, acid group and sometimes even a sulfuratom.

As for example, compounds like phenylthiomethyl pyridine, Diclonixin,Anpirtoline and Pyridyl carbothiolate contain a pyridyl moiety whereasAnpirtoline and Pyridyl Carbothiolate contain sulfur attached directlyto the pyridyl moiety. While looking for some novel anti-inflammatorycompound we propose to keep the basic pyridyl moiety intact with changein derivatization pattern on the sulfur and the carboxylic acid group.

From the existing literature it is evident that the compound of interesti.e. the most active compound RS—Z (3-(Pyridin-2-yl sulfanyl)-propionicacid pentyl ester (Ia)) is a new compound not prepared or reportedbefore. Similar compounds or compounds bearing at least thiopyridylsubstructure reported in literature are also limited. Followingcompounds have similarity to the compound of interest here.

But none of these compounds are reported to have any biologicalactivity. Compound (A), i.e. 3-(2-pyridylthio) propionic acid ethylester has been reported during the course of study on aromaticsubstitution (Rossi R A, Pierini A B, Santiago A N, NucleophilicAromatic Substitution Organic Reactions 1999, 54, Hoboken, N.J., US),addition reaction of electrogenerated thiolate anion to olefin(Niyazymbetov M E, Konyushkin L D, Niyazymbetova Z I, Litvinov V P,Petrosyan V A. Electrocatalytic addition of thiols to activated olefins.Khimicheskaya, 1991, 260), syn-elimination (Crich D, Lim L B L, J. Chem.Research (S), 1987, 353) and study of SRN1 reaction (Beugelmans R,Bois-Choussy M, Boudet B, Etude des reactions de s_(rn)1—parte 10:Action de sulfanions sur les halogenures d'aryle fonctionnalises.Synthese directe de benzothiophenes et thienopyridines Tetrahedron,1983, 39, 4153). Compound (B), i.e. 5-(2-pyridylthio) pentanoic acidmethyl ester is reported in a study on radical reactions (Barton H R,Bridon D, Fernandez-Picot I, Zard S Z, Tetrahedron, 1987, 43, 2733).Compound (C), i.e. 4-(2-pyridylthio) butanoic acid ethyl ester isreported in a study on reaction of 2-trimethylsilylmethylthiopyridinewith alkene (Kohra S, Ueda H, Tominaga Y. Reaction of2-trimethylsilylmethylthiopyridine promoted by a fluoride ion: the firstexample of generation of 2-pyridylthiomethylcarbanion Heterocycles,1993, 36, 1497). It was reported some time back that fewanti-inflammatory compounds of type phenylthiomethyl pyridine possessbiological results (Haviv F, DeNet R W, Michaels R J, Ratajczyk J D,Carter G W, Young P R. 2-[(Phenylthio)methyl]pyridine derivatives: newanti-inflammatory agents. J. Med. Chem. 1983, 26, 218).

OBJECTIVE OF THE INVENTION

The main objective of the present invention is to provide compounds ofgeneral formula 1 having anti-inflammatory activity.

Another object of the present invention is to screen compounds ofgeneral formula 1 for their anti-inflammatory activity in in-vitrocell-based assay system.

Yet another object of the present invention is to provide most activecompound of formula 1a from the series of the evaluated antiinflammatory compounds.

Still another object of the present invention is to determine the IC₅₀values for the inhibition of ICAM-1 and neutrophil adhesion by antiinflammatory compounds for functional correlation of cytokine-inducedexpression by the novel anti-inflammatory compounds.

Still another object of the present invention is to evaluate in-vivoefficacy of the selected most active novel anti-inflammatory compound offormula 1a in the mice model of LPS induced acute lung injury.

SUMMARY OF THE INVENTION

Accordingly, present invention provides compound of general formula 1

-   -   wherein    -   R₁=H;    -   R₂=H, COOMe;    -   R₃=(CH2)_(n)COOR₄;    -   R₄=C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₁;    -   n=1-7.

In an embodiment of the present invention, representative compounds ofgeneral formula 1 are:

-   3-(2-pyridylthio)propionic acid butyl ester (RS20);

-   3-(2-pyridylthio)propionic acid pentyl ester (RS Z);

-   7-(2-pyridylthio)heptanoic acid ethyl ester (RS 32);

-   3-(2-pyridylthio)propionic acid cyclohexyl ester (RS 21);

-   2-(2-pyridylthio)-hexanedioic acid 6-cyclohexyl ester 1-methyl ester    (AD 21);

-   2-(2-pyridylthio)-hexanedioic acid 1-methyl ester 6-propyl ester (AD    20);

-   2-(2-pyridylthio)-hexanedioic acid-1-methyl ester 6-pentyl ester (AD    Z);

-   2-(2-pyridylthio)-decanedioic acid-10-ethyl ester 1-methyl ester (AD    32).

In yet another embodiment of the present invention, said compounds areuseful as anti-inflammatory agent.

In yet another embodiment of the present invention, process for thepreparation of compound of general formula 1 comprising the steps of:

-   -   i. providing barton ester of formula IV by known method;

-   -   -   Wherein R=—CH₂CH₂COOC₄H₉, —CH₂CH₂COOC₅H₁₁, (CH₂)₆COOEt,            —CH₂CH₂COOC₆H₁₁, —CH(COOMe)(CH₂)₃COOC₆H₁₁,            —CH(COOMe)(CH₂)₃COOC₃H₇, —CH(COOMe)(CH₂)₃COOC₅H₁₁,            —CH(COOMe)(CH₂)7COOC₂H₅.

    -   ii. diluting barton ester as provided in step (i) with solvent        up to 25 ml;

    -   iii. irradiating the diluted barton ester under sun light at        temperature in the range of 25 to 30° C. for period in the range        of 15 to 20 minutes;

    -   iv. optionally irradiating the diluted barton ester with olefin        under sun light at temperature in the range of 25 to 30° C. for        period in the range of 15 to 20 minutes;

    -   v. removing the solvent from the irradiated solution as obtained        in step (iv) under reduced pressure in the range of 30 to 50        millibar to obtain an oily crude product;

    -   vi. purifying crude product as obtained in step (v) by        preparative TLC to obtain the compound of general formula 1.

In yet another embodiment of the present invention, solvent used isselected from dry and degassed benzene or CH₂Cl₂.

In yet another embodiment of the present invention, olefin used ismethyl acrylate.

In yet another embodiment of the present invention, electric bulb of 200watt can be used to irradiate the solution for period in the range of 8to 10 hrs.

In yet another embodiment of the present invention, said compound isprepared from the O-acyl derivative of N-hydroxy-2-thiopyridone preparedfrom mono pentyl ester derivative of butane dioic acid.

In yet another embodiment of the present invention, said compoundsexhibiting inhibition of the LPS induced ICAM-1 (Intercellular celladhesion molecule-1) expression and neutrophil adhesion on humanendothelial cells with IC₅₀ in the range of 50±0.84 to 178±0.81 μM and61±0.84 to 94±0.92 μM respectively.

In yet another embodiment of the present invention, compound RS—Zexhibiting reduction of the neutrophil influx in the lungs in a micemodel of acute lung injury at doses of 0.1, 1.0 and 10 mg/kg body weighti.p. and attenuation of the LPS-induced lung injury in mice at doses of0.1, 1.0 and 10 mg/kg body weight i.p.

In an embodiment of the present invention, bioactivity of the compoundRS—Z is characterized by known biological assays selected from the groupconsisting of anti-inflammatory and cytotoxicity assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Represent scheme for the preparation of compound of generalformula 1.

FIG. 1.1 (Synthetic scheme of RS—Z (Formula 1a).

FIG. 1.2 (Synthetic scheme of RS-20).

FIG. 1.3 (Synthetic scheme of RS-21)

FIG. 1.4 (Synthetic scheme of RS-32)

FIG. 1.5 (Synthetic scheme of AD-Z, side product is RS—Z)

FIG. 1.6 (Synthetic scheme of AD-20, RS-20 is side product)

FIG. 1.7 (Synthetic scheme of AD-21, side product RS-21)

FIG. 1.8 (Synthetic scheme of AD-32, side product RS-32) For first fourcompounds, preparation of Barton ester as well as its photolysis isshown and for the rest photolysis of Barton ester with methyl acrylateis shown only, its preparation is same as first four).

FIG. 2A. Total cell counts in the BALF (bronchoalveolar lavage fluid)supernatant in mice. Post-treatment with IP (intraperitoneal)administration of RS—Z dose-dependently reduced the increased cellcounts induced by LPS. The data are mean±S.E.M. of six mice for eachgroup.* P<0.001 vs. Saline aerosol/vehicle IP; P<0.001 vs. LPSaerosol/vehicle IP.

FIG. 2B. Differential cell count in the BALF supernatant in mice.Post-treatment with RS—Z dose-dependently reduced the increasedneutrophil count induced by LPS. The data are mean±S.E.M. of six micefor each group. *P<0.001 vs. Saline aerosol/vehicle IP; P<0.001 vs. LPSaerosol/vehicle IP.

FIG. 3: Effects of RS—Z on the injury score in the lung tissue in miceof acute lung injury induced by lipopolysaccharide (LPS) aerosolinhalation. Post treatment with RS—Z dose-dependently reduced theincreased injury induced by LPS. The data are mean±S.E.M. of six micefor each group. * P<0.0001 vs. Saline aerosol/vehicle IP; P<0.0001 vs.LPS aerosol/vehicle IP.

DETAIL DESCRIPTION OF THE INVENTION

Present invention provides anti-inflammatory Pyridin-2-yl sulfanyl acidester compounds of general formula 1

whereinR₁=H;R₂=H, COOMe;R₃=(CH₂)_(n)COOR₄;R₄=C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₁;n=1-7.

Chemically these compounds are classified as Pyridin-2-yl sulfanyl acidester or Carboxylic acid, n-(2-pyridinylthio)-, alkyl ester. These typesof compounds are prepared from O-acyl derivative ofN-Hydroxy-2-thiopyridone (popularly called as Barton ester). The Bartonester of interest is irradiated under Sun light or under visible lightfor a specified period of time to get the desired product. Compounds ofinterest may also be prepared:

-   i. from Barton ester under thermal condition;-   ii. from a pure isolated Barton ester through photolysis under    sunlight/normal light or from the Barton ester prepared in situ    without further treatment or isolation;-   iii. from the photolysis reaction of Barton ester alone or in    presence of some olefinic trap as detailed elsewhere.

The compounds of interest may be purified through chromatographic meansand isolated from the photolysis reaction mixture of the Barton ester.The derivatives of Pyridin-2-yl sulfanyl acid esters were synthesizedand characterized. These compounds were further screened for theiranti-inflammatory activity with respect to inhibition of adhesion ofneutrophils, isolated from human peripheral blood, onto the surface ofhuman umbilical vein endothelial cells (HUVEC) as a result of inhibitionof the cytokine-stimulated expression of ICAM-1. The IC₅₀ values for thecompounds (Table 1) with respect to their inhibition of ICAM-1expression and neutrophil adhesion on endothelial cells were determined.The compounds were found to be non-toxic to endothelial cells, however,the maximum tolerable dose for each compound was found to be different.

TABLE 1 Screening data of the derivatives synthesized. Neutrophil Max.ICAM-1 Adhesion tolerable inhibition IC₅₀ Inhibition IC₅₀ dose (μM) (μM)(μM)* RS-21 135 ± 0.82 not significant 250 RS-32  97 ± 0.63 94 ± 0.92500 RS-20  79 ± 0.73 85 ± 0.94 500 RS—Z  50 ± 0.84 61 ± 0.84 500 AD-21117 ± 0.92 not significant 250 AD-20 178 ± 0.81 not significant 250AD-32 100 ± 0.96 not significant 250 AD—Z  85 ± 1.52 not significant 250NAC 6.68 mM ± 1.23    not significant 20 mM (N-acetyl cysteine)(reference compound) *Concentration of compounds where >95% cells areviable

The compound designated as RS—Z (Formula 1a) was found to be most potentanti inflammatory compound of the series with lowest IC₅₀ values forICAM-1 and neutrophil adhesion inhibition. This compound was also foundto have in-vitro maximum tolerable dose of 500 μM on human umbilicalvein endothelial cell (HUVEC).

Characterization of the anti-inflammatory lead compound of formula 1awas carried out by known analytical methods such as infrared, mass andnuclear magnetic resonance spectroscopy and biological assays such asanti-inflammatory assay and cytotoxicity assay having followingcharacteristics:

IR (CHCl3): (cm⁻¹) 3060, 2960, 2853, 1780, 1733, 1452, 1415, 1123, 1043,987;

MS (m/z) %: 253.8 (M+1)

¹H NMR (CDCl₃): (∂ ppm) 8.50 (ddd, J=4.9, 1.8, 1 Hz, 1H), 7.48 (ddd,J=8, 7.8, 1.8 Hz, 1H), 7.17 (td, J=8, 4.5 Hz, 1H), 7.00 (ddd, J=7.8,4.9, 1 Hz, 1H), 4.10 (t, J=7.2 Hz, 2H), 3.15 (t, J=7 Hz, 2H), 2.30 (t,J=7 Hz, 2H), 1.70-1.20 (m, 6H), 0.90 (t, J=7 Hz, 3H);

¹³C NMR (CDCl₃): (∂ ppm) 173.00, 159.23, 148.17, 136.27, 122.30, 119.17,60.75, 33.15, 29.10, 26.12, 22.65, 20.37, 14.75

The said compound of formula 1a causes

-   -   a) Inhibition of the LPS-induced ICAM-1 expression on human        endothelial cells and has IC₅₀ of 50 μM.    -   b) Inhibition of the LPS-induced neutrophil adhesion on the        surface of human endothelial cells with IC₅₀ of 61 μM.    -   c) Reduction of the neutrophil influx in the lungs in a mice        model of acute lung injury at doses of 0.1, 1.0 and 10 mg/kg        body weight., i.p.    -   d) Attenuation of the LPS-induced lung injury in mice at doses        of 0.1, 1.0 and 10 mg/kg body weight., i.p.

TABLE 2 Comparison of RS—Z with known compounds in acute lung Injurymodel. Efficacious Dose Compound (mg/kg) Class of compound Reference 1.RS—Z (3-(Pyridin-2-yl  10 mg/kg Pyridin-2-yl sulfanyl —sulfanyl)-propionic acid pentyl acid ester ester 2. Dexamethasone  3mg/kg Glucocorticoid Clin. Hemorheol. microcirc., 2009, 41(2), 117-25 3.N-acetyl Cysteine 150 mg/kg Antioxidant and free- Clinical andExperimental radical scavenger pharmacology and Physiology, 2006,January-February, 33(1-2), 33-40 4. Rolipram  5 mg/kg Phosphodiesterase4 Am J Respir. Cell mol Biol., inhibitor 1998, 18(3), 411-20 5.Water-soluble Vitamin E  10 mg/kg Antioxidant Clinical and Experimentalderivative(α-D-glucopyranosyl) pharmacology and methyl-2,5,7,8-Physiology, 2004, 31(4), (tetramethyl)chroman-6-ol) 226-230 6.Thalidomide 100 mg/kg Sedative J. Biomed. Sci., 2004, 11,(α-N-phthalimidoglutarimide) 591-598

The derivatives of Pyridin-2-yl sulfanyl acid esters were characterizedby IR, NMR and mass spectroscopy prior to screening for theiranti-inflammatory activity with respect to inhibition of adhesion ofneutrophils, isolated from human peripheral blood, onto the surface ofhuman umbilical vein endothelial cells (HUVECs) as a result ofinhibition of the cytokine-stimulated expression of ICAM-1.

The compounds were found to be non-toxic to endothelial cells, however,the maximum tolerable dose for each compound was found to be different.The IC_(50 values) for the compounds (Table 1) with respect to theirinhibition of ICAM-1 expression and neutrophil adhesion on endothelialcells were determined.

Biological Activity of the Compound

Cell-cell interactions that are critical for normal hemostasis, immunesurveillance, and vascular wall integrity are mediated by glycoproteinsknown as cell adhesion molecules (CAMs). These protein molecules mediateleukocyte-endothelial cell interactions that occur in all segments ofthe microvasculature under certain physiological (eg, hemostasis) andpathological (eg, inflammation) conditions (Muller A M et al, 2002). Thebidirectional interactions between leukocytes and endothelial cells areinfluenced by cytokines such tumor necrosis factor (TNF-α), IL-1β andgram-negative bacterial polysaccharide (LPS) that drastically increasethe expression of cell adhesion molecules on the surface of theendothelium (Sato N et al, 2000). Aberrant interaction between theleukocyte and the endothelial cell (EC) results in the uncontrolledinflammation leading to various inflammatory disorders. One of thepromising therapeutic target to control the dysregulatedleukocyte-endothelial interaction would be to modulate thecytokine-induced expression of cell molecules (Bhatia M, 1998). In orderto treat the deleterious inflammatory responses in human diseases,anti-inflammatory agents are extensively used clinically. However,anti-inflammatory drugs such as NSAIDs (Non-steroidal anti-inflammatorydrugs), corticosteroids and chemotherapeutic agents have severe sideeffects. Therefore, there is an unmet therapeutic need to develop moreselective and safe drugs to treat inflammatory disorders.

In search of a new anti-inflammatory drug with negligible side effects,the novel synthesized compounds were screened for theiranti-inflammatory activity with respect to inhibition of adhesion ofneutrophils isolated from human peripheral blood, onto the surface ofhuman umbilical vein endothelial cells (HUVECs) as a result ofinhibition of the cytokine-stimulated expression of ICAM-1.

The present invention showed that the compounds are not toxic toendothelial cells, however, the maximum tolerable dose for each compoundis different. The IC₅₀ values for the compounds (Table 1) with respectto their inhibition of ICAM-1 expression and neutrophil adhesion onendothelial cells were calculated. RS—Z (formula 1a) showed the lowestIC₅₀ values of 50 μM for ICAM-1 inhibition and 61 μM for neutrophiladhesion inhibition.

Therefore, the present invention showed that the most active compound ofthe series was 3-(Pyridin-2-yl sulfanyl)-propionic acid pentyl ester(RS—Z, formula 1a) that could inhibit both the expression of celladhesion molecule ICAM-1 (intercellular adhesion molecule-1) (IC₅₀=50μM) and the neutrophil adhesion onto the surface of human endothelialcells (IC₅₀=61 μM).

The compound RS—Z was tested for in-vivo efficacy in a mice model. Micewere sensitized with aerosol inhalation of LPS to develop thecharacteristic features of acute lung injury such as massiveinfiltration of neutrophils into the lung leading to alveolar damage.These features were characterized by determining the lung injury scoreusing the known scoring methods from the H & E (hematoxylin & eosin)stained slides of the excised lung tissue, total and differential cellcount was performed in the BALF (bronchoalveolar lavage fluid) todetermine the infiltration status.

The present invention showed that the novel compound RS—Z couldsignificantly alleviate pulmonary neutrophil infiltration in mice modelof acute lung injury induced by bacterial polysaccharide at a dose of 10mg/kg body weight administered intraperitoneally. The present inventionshowed that RS—Z significantly reduced the lung injury caused bybacterial polysaccharide challenge at a dose of 10 mg/kg body weight.

EXAMPLES

Following examples are given by way of illustration therefore should notbe construed to limit the scope of the present invention.

Example 1 Step 1 Preparation of Butane Dioic Acid Mono Alkyl Ester (II)

3 mmol (300 mg) of succinic anhydride was dissolved in 5 ml ofdichloroethane in a 50 ml conical flask. To the solution was added 3mmol of an alkanol followed by 151 mg (1.5 mmol) of dry triethylamine.The flask was loosely stoppered with cotton plug and irradiated in amicrowave oven at 80% power (480 watt) for 3 minutes. The reactionmixture was cooled and diluted with 15 ml of ethyl acetate. The ethylacetate extract was washed 3 times with 30 ml of water each. The organiclayer was dried over anhydrous sodium sulfate and evaporated underreduced pressure. The crude product was purified by preparative TLCusing 1:4 (ethyl acetate:hexane) solvent system to get around 75% ofpure butanedioic acid mono alkyl ester (II) as gum. The product wascharacterized in a usual way by recording IR, NMR and MS spectra.

R may be alkyl group like methyl, ethyl, propyl, butyl, pentyl, hexyletc or substituted alkyl group, cycloalkyl, benzyl, naphthyl etc.

Step 2 Preparation of Alkane Dioic Acid Mono Alkyl Ester (III)

Alkane dioic acid mono alkyl ester was prepared from alkane dioic acidthrough steps (a) and (b) as discussed below:

(a) Preparation of the Diester

To a solution of 3 mmol of the alkane dioic acid in 5 ml of absolutealkanol in a 50 ml conical flask was added two drops of conc. H₂SO₄acid. The flask was loosely stoppered with a cotton plug and irradiatedin a microwave oven at power 60% (360 watt) for four minutes. Thereaction mixture was cooled and diluted with 15 ml of ethyl acetate. Theethyl acetate layer was washed with aqueous sodium bicarbonate solutionto remove the unreacted diacid. The ethyl acetate layer was finallywashed with water and dried over anhydrous sodium sulfate. The solventwas removed by distillation under reduced pressure in a rotaryevaporator to get 75% of the diester as oil. The structure of the alkanedioic acid diester was confirmed by recording IR, NMR and MS spectra.

(b) Selective Hydrolysis of the Dioic Acid Dialkyl Ester to Mono Ester(III)

To a three necked 250 ml R.B. flask fitted with mercury sealedmechanical stirrer, a dropping funnel and a reflux condenser with aCaCl₂ guard tube on its top was added 0.01 mol of the dialkyl ester ofalkane dioic acid in 10 ml absolute ethanol with continuous stirring atroom temperature. A solution of 0.01 mol (560 mg) of potassium hydroxidein 10 ml absolute ethanol was added drop wise over a period of one hourunder stirring condition. During addition of alkali, white crystallineprecipitate was observed to form. After complete addition of potassiumhydroxide, stirring was continued for further two hours and the reactionmixture was kept overnight at RT. Excess alcohol was distilled off underreduced pressure, the residue was diluted with water (10 ml) and theaqueous layer was extracted with ether (3×10 ml) to remove the unchangeddiester from potassium salt. The potassium salt in the aqueous layer wascooled to 3° C., and acidified by adding 4 ml of 6N HCl drop wise over aperiod of 30 minutes. The aqueous layer was extracted with 3×10 ml ofether and then the ether layer was dried over anhydrous Na₂SO₄. Excessether was evaporated on a water bath. The crude residue was purified bycolumn chromatography to obtain about 48% of pure acid ester (III) asgum. The acid ester (III) was characterized as usual way.

Example 2 Preparation of Butanedioic Acid n-Pentyl Monoester (IIa)

Butanedioic acid n-pentyl monoester (IIa) was prepared by following thesame procedure as described for the preparation of (II) (step 1).Amounts of substrate and reagents used in the reaction are mentionedbelow. Product (IIa) was isolated as gum after purification by usingcolumn chromatography.

Succinic anhydride: 300 mg=3 mmol

n-petanol: 264 mg=3 mmol

Dry triethylamine: 151 mg=1.5 mmol

Dichloroethane: 5 ml

MW power: 70% (480 watt)

Time: 3 minutes

Product (IIa) was characterized as follows:

1H NMR (CDCl₃): (δ ppm) 10.10 (brs, 1H), 4.20 (t, J=7.2 Hz, 2H), 2.60(t, J=7 Hz, 4H), 1.30-1.60 (overlapping multiplet, 6H) and 0.93 (m, 3H).

IR (CHCl₃): (cm⁻¹) 340, 2980, 1730, 1710, 1460, 1342, 1220, 1180, 1120,1029.

MS (m/z) %: 187 (M+1)

Example 3 Preparation of Intermediate Preparation of Butane Dioic AcidButyl Monoester (IIb)

Butane diooic acid butyl monoester (IIb) was prepared following theprocedure described for preparation of (II). (Step 1) Amounts ofsubstrate and reagents used in the reaction are as mentioned bellow. Thecrude product was purified by using column chromatography.

Succinic anhydride: 300 mg=3 mmol

n-butanol: 222 mg=3 mmol

Dry triethylamine (TEA): 151 mg=1.5 mmol

Dichloromethane: 3 ml

MW power: 80% (480) watt

Time: 3 minutes

Yield: 365 mg=70%

Product (IIb) was characterized as follows:

¹H NMR (CDCl₃): (δ ppm) 10.20 (s, H), 4.20 (t, J=7 Hz, 2H), 2.60 (m,4H), 1.60-1.30 (m, 4H), 0.92 (t, J=7 Hz, 3H).

IR (CHCl₃): (cm⁻¹) 3455, 2981, 2942, 1733, 1710, 1462, 1447, 1375, 1349,1220, 1147, 1077, 1029.

MS (m/z) %: 173 (M+1)

Example 4 Preparation of Intermediate Preparation of Octane Dioic AcidMono Ethyl Ester

Octane dioic acid monoethyl ester was prepared from octane dioic acidthrough steps (a) and (b) as discussed in Example 1, Step 2 above.

(a) Preparation of Octane Dioic Acid Diethyl Ester

Octane dioic acid diethyl ester was prepared by following the proceduredescribed for the preparation of diester in example 2 (a). The diesterwas isolated as gum in 70% yield. The amounts of substrate and reagentsused in the reaction are mentioned below.

Octane dioic acid: 522 mg=3 mmol

Absolute ethanol: 5 ml

Conc. H₂SO₄ acid: 2 drops

MW power: 60%

Time: 3 minutes

Yield: 483 mg=70%

Octane Dioic Acid Diethyl Ester

The diester was characterized as follows.

¹H NMR (CDCl₃): (δ ppm) 4.10 (q, J=7 Hz, 4H), 2.50 (t, J=7 Hz, 4H),1.50-1.60 (m, 4H), 1.30-1.10 (m, 10H).

IR (CHCl₃): (cm⁻¹) 2980, 2945, 2870, 1712, 1451, 1412, 1220, 1157, 1020,928, 770.

MS (m/z) %: 231 (M+1).

(b) Selective Hydrolysis of Diethyl Ester of Octane Dioic Acid to GetOctane Dioic Acid Mono Ethyl Ester (IIIa)

Selective hydrolysis of diethyl ester of octane dioic acid to get acidester (IIIa) was done by following the procedure described for theselective hydrolysis of ester in example 2 (b). The product (IIIa) wasisolated as gum in 49% yield after purification by columnchromatography.

The acid ester (IIIa) was characterized as follows.

¹H NMR (CDCl₃): (δ ppm) 9.30 (br, s, H), 4.10 (q, J=6 Hz, 2H), 2.30-2.50(m, 4H), 1.60 (m, 4H), 1.30-1.10 (overlapping multiplet, 7H).

IR (CHCl3): (cm⁻¹) 3022, 2970, 2942, 2882, 1730, 1717, 1460, 1380, 1217,1180, 1060, 940.

MS (m/z) %: 203 (M+1).

Example 5 Preparation of Intermediate Preparation of Pentanedioic AcidMonopropyl Ester (IIIb)

Pentane dioic acid mono propyl ester was prepared from pentane dioicacid through two steps as described below.

(a) Preparation of Pentane Dioic Acid Dipropyl Ester

To a solution of 396 mg (3 mmol) of pentane dioic acid in 3 ml ofn-propanol in a 50 ml conical flask was added 2 drops of conc. H₂SO₄acid. The flask was loosely stoppered by cotton and subjected toirradiation in a microwave oven at power 60% (360 watt) for 4 minutes.The reaction mixture was cooled and diluted with 15 ml ethyl acetate.The ethyl acetate extract was washed with aq. sodium bicarbonatesolution to remove the unreacted diacid. The ethyl acetate layer wasfinally washed with water and dried over anhydrous sodium sulfate. Thesolvent was removed by distillation under reduced pressure in rotaryevaporator. The oily crude so obtained was purified by using columnchromatography. The diester was recovered as gum in 70% yield.

Pentane Dioic Acid Dipropyl Ester

The diester was characterized as follows:

¹H NMR (CDCl₃): (δ ppm) 4.15 (t, J=7 Hz, 4H), 2.60 (t, J=7 Hz, 4H), 1.60(t, J=7 Hz, 4H), 1.15 (m, J=7 Hz, 4H), 0.90 (t, J=7 Hz, 6H).

IR (CHCl₃): (cm⁻¹) 2960, 2910, 2830, 1730, 1448, 1370, 1352, 1265, 1210,1160, 1030.

ms (m/z) %: 202 (M+1).

(b) Selective Hydrolysis of Pentanedioic Acid Dipropyl Ester

To a doubled necked 250 ml Round Bottom (R.B.) flask fitted with adropping funnel and a reflux condenser with a CaCl₂ guard tube on itstop was added 0.010 mol of the dipropyl ester of the pentanedioic acidin 10 ml of absolute ethanol at room temperature. A solution of 560 mg(0.01 mmol) of potassium hydroxide in 10 ml of absolute ethanol wasadded drop wise over a period of one hour under continuous stirring.During addition of the alkali, white crystalline precipitate wasobserved to form. After complete addition of potassium hydroxide,stirring was continued for another two hours and the reaction mixturewas kept overnight at room temperature. Excess alcohol was distilled offunder reduced pressure, the residue was diluted with water (10 ml) andthe aqueous layer was extracted with ether (3×10 ml) to remove theunchanged ester from potassium salt. The potassium salt in the aqueouslayer was then cooled to 5° C., and acidified by adding 4 ml of 6N HClacid drop wise over a period of 30 minutes. The aqueous layer wasextracted with 3×10 ml of ethyl acetate and the ethyl acetate layer wasdried over anhydrous Na₂SO₄. Excess ethyl acetate was evaporated underreduced pressure in rotary evaporator. The oily crude was purified bycolumn chromatography to obtain 49% of pure acid ester (15b) as gum.

The acid ester (IIIb) was characterized as follows:

¹H NMR (CDCl₃): (δ ppm) 10.30 (br, s, H), 4.20 (t, J=7 Hz, 2H), 2.50 (t,J=7 Hz, 4H), 1.60 (m, 2H), 1.20 (m, 2H), 0.90 (t, J=7 Hz, 3H).

IR (CHCl₃): (cm⁻¹) 3420, 2960, 2940, 1732, 1718, 1420, 1395, 1215, 1185,1062, 945.

MS (m/z) %: 180 (M+1).

Example 6 Preparation of Intermediate Preparation of Hexane Dioic AcidMono Ethyl Ester (IIIc)

Hexane dioic acid monoethyl ester was prepared from hexane dioic acidthrough steps (a) and (b) discussed above in example 1, step 2 for thepreparation of (III).

(a) Preparation of Diethyl Ester of Hexane Dioic Acid

Diethyl ester of hexane dioic acid was prepared by following the sameprocedure as described for the preparation of the diester in Example 2(a). Compound was isolated as oil in 75% yield after purification bycolumn chromatography. Amounts of substrate and reagents used in thereaction are as mentioned below.

Hexane dioic acid: 439 mg=3 mmol

Absolute ethanol: 5 ml

Conc. H₂SO₄ acid: 2 drops

M.W. power: 80%

Time: 3 minutes

Ethyl Diester of Hexanedioic Acid

The diester was characterized as follows.

Yield: 75%

¹H NMR (CDCl₃): (δ ppm) 4.15 (q, J=7 Hz, 4H), 2.60 (t, J=7 Hz, 4H), 1.60(m, 4H), 1.10 (t, J=5 Hz, 6H).

IR (CHCl₃): (Cm⁻¹) 2980, 2939, 2612, 1730, 1458, 1373, 1349, 1265, 1214,1162, 1033.

MS (m/z) %: 203 (M+1)

(B) Selective Hydrolysis of Diethyl Ester of Hexane Dioic Acid to GetHexane Dioic Acid Mono Ethyl Ester (IIIc)

Selective hydrolysis of diethyl ester to get acid ester (IIIc) was doneby following the procedure described for the selective hydrolysis ofdiethyl ester in example 2 (b). The product (IIIc) was isolated as oilin 52% yield after purification by column chromatography.

The acid ester (IIIc) was characterized as follows.

¹H NMR (CDCl₃): (δ ppm) 9.60 (br, s, H), 4.10 (q, J=3 Hz, 2H), 2.60 (t,J=7 Hz, 4H), 1.60 (m, 4H) and 1.00 (t, J=7 Hz, 3H).

IR (CHCl₃): (cm⁻¹) 3024, 2980, 2939, 2882, 1730, 1717, 1414, 1396, 1362,1217, 1173, 1060, 940, 759, 668.

ms (m/z) %: 174 (M+1).

Example 7 Preparation of Intermediate Preparation of O-Acyl Derivativeof N-Hydroxy-2-thiopyridone (Barton Ester) (IV)

Ester or more strictly the anhydride derivative ofN-Hydroxy-2-thiopyridone with carboxylic acids (O-acyl derivative) ispopularly called as Barton Ester. Barton Esters can be prepared byeither acid chloride method or by direct coupling method of acid withN-Hydroxy-2-thiopyridone in presence of dicyclohexyl carbodiimide (DCC).As Barton Esters are sensitive to heat, light and moisture at normalconditions, they are always prepared under cover of aluminum foil in adry and inert atmosphere immediately before their use and in most casesBarton Esters are neither isolated nor characterized.

DCC Method:

A double necked round bottom flask fitted with an inert gas inlet and astopper, was covered with a sheet of aluminum foil. A solution of 1 mmolof the acid in 25 ml of dry solvent (Benzene, dichloromethane, Tolueneetc.) was placed in the R.B. To the stirred solution, 0.99 mmol (125 mg)of N-hydroxy-2-thiopyridone was added, followed by 0.99 mmol of DCC atthe same temperature. Progress of the reaction was monitored by TLC. Thereaction mixture was quickly filtered by passing through a bed of silicagel to separate the precipitate of DCC-urea and the filtrate (BartonEster) was used for the next reaction without further isolation.

Acid Chloride Method:

A solution of 1 mmol of N-hydroxy-2-thiopyridone in 20 ml dry benzene ordichloromethane was placed in a double necked R.B. covered with analuminium foil under inert atmosphere. A solution of 0.99 mmol of theacid chloride in 20 ml of dry benzene or dichloromethane was added veryslowly with stirring followed by 0.1 ml of dry pyridine at 5-10° C.Stirring was continued till the completion of the reaction. Reaction wasmonitored by TLC (2:1 hexane:ethyl acetate). A single yellow spot on theTLC plate confirmed the completion of the reaction. Barton Ester soobtained was utilized in the next step without any purification andcharacterization.

General Procedure for the Preparation of Acid Chloride

Carboxylic acid chlorides were prepared immediately prior to their useby either of the methods mentioned below and used in the next reactionwithout isolation and characterization. (a) Thionyl chloride method (b)Phosphorous trichloride method (c) Oxalyl chloride method.

Example 8 Preparation of Intermediate Preparation of O-acyl Derivativeof N-Hydroxy-2-thiopyridone (Barton Ester) (IVa) with Butane Dioic Acidn-pentyl Monoester (IIa)

Succinic acid pentyl ester-2-thiopyridine-1-yl ester

The required ester was prepared by DCC method as described before inexample 7 for the preparation of the compound type (IV).

Butane dioic acid mono pentyl ester: 188 mg=1 mmol.

N-hydroxy-2-thiopyridone: 125 mg=0.99 mmol.

1,3-dicyclohexylcarbodiimide (DCC): 204 mg=0.99 mmol.

Dry benzene: 20 ml

Time: 2.10 hrs

Temperature: 20° C. (room temp.)

Yield: 100% on TLC

Example 9 Preparation of Intermediate Preparation of O-acyl Derivativeof N-Hydroxy-2-thiopyridone (Barton Ester) (IVb) with Octane Dioic AcidMono Ethyl Ester (IIIa)

Octane Dioic Acid Ethyl Ester-2-thiopyridine-1-yl Ester

The required ester was prepared by DCC method as described before inexample 8 for the preparation of the compound type (IV).

Octane dioic acid ethyl monoester: 202 mg=1 mmol

N-hydroxy-2-thiopyridine: 125 mg=0.99 mmol

1,3-dicyclohexyl carbodiimide (DCC): 204 mg=0.99 mmol.

Dry benzene: 20 ml

Time: 2 hrs 30 min.

Temperature: 25° C. (room temp.)

Yield: 100% (on TLC)

Example 10 General Procedure for the Photolysis ofN-Hydroxy-2-thiopyridone Ester (Barton Ester) (IV) Blank Photolysis toGet Compound of Type (I)

The O-acyl derivatives of N-hydroxy-2-thiopyridone (Barton ester) (IV)were prepared in situ either by following acid chloride method or by DCCmethod as above. After removing the DCC-urea by filtration, the filtratecontaining crude Barton Ester (1-0.9 mmol) was placed in a 100 ml roundbottom flask and diluted with dry and degassed benzene or CH₂Cl₂ up to25 ml. The ester solution was irradiated under sun light at temperaturein the range of 15 to 30° C. Irradiation of the Barton ester under anormal 200 watt bulb kept at a distance 30 cm from the reaction flaskalso gave almost same result. Under the sun light the reaction requires15 to 20 minutes for completion of photolysis whereas time required forphotolysis under electric bulb of 200 watts is in the range of 8-10 hrs.The progress of the reaction was monitored by TLC (1:2 EA:hexane).Disappearance of the characteristic yellow colour of the reactionmixture as well as the yellow spot on the TLC indicated the completionof the photolysis reaction. On completion, the solvent of the reactionmixture was distilled off under reduced pressure in the range of 30 to50 millibar to obtain an oily crude product in general. Purification bypreparative TLC gave the photolysis products in different amount ofyields. All the products were identified by spectroscopic analysis suchas IR, NMR and MS. Blank photolysis of compound in structure (IV) is arearrangement reaction as shown below.

Example 11 Preparation of Product RS—Z Blank Photolysis of Succinic AcidPentyl ester-2-thiopyridine-1-yl Ester (IVA)

Photolysis of the Barton ester (IVa) i.e. Succinic acid pentylester-2-thiopyridine-1-yl ester under sun light as per the procedurestated in example 10 above was done.

Butane dioic acid pentyl monoester: =188 mg=1 mmol

N-hydroxy-2-thiopyridone: =124 mg=0.99 mmol

1,3,-Dicyclohexyl carbodiimide: =204 mg=0.99 mmol

Dry dichloromethane: =25 ml

Yield of Barton ester: =100% (on TLC)

Time for photolysis: =20 min

Temperature: =25° C. (room temp.)

After completion of photolysis, solvent was removed under reducedpressure and the oily crude was purified by preparative TLC (1:5EA:Hexane). The pure products so obtained were characterized as follows:

3-(2-pyridylthio)propionic acid pentyl ester

Yield: 172 mg (68%)

¹H NMR (CDCl₃): (∂ ppm) 8.50 (ddd, J=4.9, 1.8, 1 Hz, 1H), 7.48 (ddd,J=8, 7.8, 1.8 Hz, 1H), 7.17 (td, J=8, 4.5 Hz, 1H), 7.00 (ddd, J=7.8,4.9, 1 Hz, 1H), 4.10 (t, J=7.2 Hz, 2H), 3.15 (t, J=7 Hz, 2H), 2.30 (t,J=7 Hz, 2H), 1.70-1.20 (m, 6H), 0.90 (t, J=7 Hz, 3H)

IR (CHCl3): (cm⁻¹) 3060, 2960, 2853, 1780, 1733, 1452, 1415, 1123, 1043,987.

¹³C NMR (CDCl₃): (∂ ppm) 173.00, 159.23, 148.17, 136.27, 122.30, 119.17,60.75, 33.15, 29.10, 26.12, 22.65, 20.37, 14.75

MS (m/z) %: 253.8 (M+1)

Example 12 Preparation of Product RS-32 Blank Photolysis of Octane DioicAcid Ethyl ester-2-thiopyridine-1-yl Ester (IVb)

Photolysis of the Barton ester (IVb) i.e. Octane dioic acid ethylester-2-thiopyridine-1-yl ester under sun light as per the procedurestated in example 11 above was done.

Octane dioic acid ethyl monoester: =202 mg=1 mmol

N-hydroxy-2-thiopyridone: =124 mg=0.99 mmol

1,3,-Dicyclohexyl carbodiimide: =204 mg=0.99 mmol

Dry dichloromethane: =25 ml

Yield of Barton ester: =100% (on TLC)

Time for photolysis: =18 min

Temperature: =20° C. (room temp.)

After completion of photolysis, solvent was removed under reducedpressure and the oily crude was purified by preparative TLC (1:5EtAc:Hexane). The pure products so obtained were characterized asfollows:

7-(2-pyridylthio)heptanoic acid ethyl ester

Yield: 191 mg (68%)

¹H NMR (CDCl₃): (∂ ppm) 8.39 (ddd, J=4.9, 1.8, 1 Hz, 1H), 7.40 (ddd,J=8, 7.8, 1.8 Hz, 1H), 7.16 (td, J=8, 4.5 Hz, 1H), 6.99 (ddd, J=7.8,4.9, 1 Hz, 1H), 4.10 (q, J=7 Hz, 2H), 3.15 (t, J=7 Hz, 2H), 2.40 (t, J=7Hz, 2H), 1.70-1.30 (m, 8H), 1.10 (t, J=7 Hz, 3H)

IR (CHCl₃): (cm⁻¹) 3050, 2950, 2870, 1730, 1580, 1462, 1416, 1280, 1225,1040.

¹³C NMR (CDCl₃): (∂ ppm) 173.05, 157.25, 149.60, 136.42, 122.27, 119.25,80.50, 35.82, 33.25, 31.16, 30.70, 28.65, 25.32, 14.60.

MS (m/z) %: 267.8 (M+1)

Example 13 Preparation of Product AD-Series

General Procedure for Photolysis of Barton Esters (IV) in Presence ofOlefin as Trap

O-acyl derivative of N-hydroxy-2-thiopyridone (Barton Esters, 1 mmol),prepared in situ or isolated as pure was placed in a 100 ml size roundbottomed flask covered by aluminum foil. The ester solution was dilutedup to 23 ml by adding anhydrous and digassed benzene or CH₂Cl₂. In aninert atmosphere, 5 mmol of an olefin like methyl acrylate was added tothe solution and irradiated with sunlight at room temperature of 27° C.Irradiation with light from 200 W bulb kept a distance of 30 cum fromthe reaction served the same purpose giving almost the same resultexcept slight decrease in yield. As discussed earlier, photolysis under200 W light requires much more time (8-10 hrs) as compared to photolysisunder sunlight (15 to 20 minutes). The progress of the reaction wasmonitored by TLC. Disappearance of the characteristic yellow spot ofBarton ester on TLC plate indicated the completion of the reaction. Thesolvent was removed under reduced pressure to obtain an oily crudeproduct in general. Purification by chromatographic means gave thephotolytic products in different amount of yields. All the products werecharacterized by spectroscopic analysis such as IR, NMR and MS.

Example 14 Preparation of Product AD-Z

Photolysis of Barton Esters (IVa) in Presence of Olefin as Trap

Photolysis of the Barton ester (IVa) i.e. Succinic acid pentylester-2-thiopyridine-1-yl ester in presence of methyl acrylate under sunlight as per the procedure stated in example 13 above was done.

Butane dioic acid mono pentyl ester: 188 mg=1 mmol.

N-hydroxy-2-thiopyridone: 125 mg=0.99 mmol.

1,3-dicyclohexylcarbodiimide (DCC): 204 mg=0.99 mmol.

Dry benzene: 20 ml

Time: 2.10 hrs

Temperature: 15-20° C. (room temp.).

Yield: 100% on TLC

Methylacrylate: 0.45 ml (5 mmol)

Time of photolysis: 18 min.

Yield of adduct: 170 mg (50%)

Example 15 Preparation of Product AD-32

Photolysis of Barton Esters (IVb) in Presence of Olefin as Trap

Photolysis of the Barton ester (IVb) i.e. Octane dioic acid ethylester-2-thiopyridine-1-yl ester in presence of methyl acrylate under sunlight as per the procedure stated in example 14 above was done.

Octane dioic acid ethyl monoester: =202 mg=1 mmol

N-hydroxy-2-thiopyridone: =124 mg=0.99 mmol

1,3,-Dicyclohexyl carbodiimide: =204 mg=0.99 mmol

Dry dichloromethane: =25 ml

Yield of Barton ester: =100% (on TLC)

Methylacrylate: =0.45 ml (5 mmol)

Time of photolysis: =18 min.

Temperature: =15-20° C. (room temp.)

Yield of adduct: =187 mg (53%)

Example 16

Cell Culture:

Primary endothelial cells were isolated from human umbilical cord usingmild trypsinization (Kumar, S., Arya, P., Mukherjee, C., Singh, B. K.,Singh, N., Parmar, V. S., Prasad, A. K., Ghosh, B. Novel Aromatic Esterfrom Piper longum and Its Analogues Inhibit Expression of Cell AdhesionMolecules on Endothelial Cells. Biochemistry. 2005, 44, 15944-15952).The cells were grown in M199 medium (Sigma, USA) supplemented with 15%heat inactivated fetal calf serum (Biological Industries, Israel), 2 mML-glutamine (Sigma, USA), 100 units/ml penicillin (Sigma, USA), 100μg/ml streptomycin (Sigma, USA), 0.25 μg/ml amphotericin B (Sigma, USA),endothelial cell growth factor (50 μg/ml) (Sigma, USA). At confluence,the cells were subcultured using 0.05% trypsin-0.01 M EDTA solution andwere used between passages three to four.

Example 17 Cell Viability Assay

The cytotoxicity of these compounds was analyzed by colorimetric MTT(methylthiazolydiphenyl-tetrazolium bromide, Sigma, USA) assay asdescribed (Kumar S et al, 2005). Briefly, endothelial cells were treatedwith DMSO alone (0.25%, as vehicle) or with different concentrations ofcompounds for 24 hrs. Four hrs before the end of incubation, medium wasremoved and 100 μl MTT (5 mg/ml in serum free medium) was added to eachwell. The MTT was removed after 4 hrs, cells were washed out with PBS(phosphate buffered saline, pH 7.4), and 100 μl DMSO was added to eachwell to dissolve water insoluble MTT-formazan crystals. Absorbance wasrecorded at 570 nm in an ELISA reader (Bio-Rad, Model 680, USA). Allexperiments were performed at least 3 times in triplicate wells.

Example 18 Cell-ELISA for Measurement of ICAM-1

Cell-ELISA was used for measuring the expression of ICAM-1 on surface ofendothelial cells (Kumar S et al, 2005) Endothelial cells were incubatedwith or without the test compounds at desired concentrations for therequired period, followed by treatment with LPS (1 μg/ml) (BD, USA) for16 hrs for ICAM-1 expression. The cells were fixed with 1.0%glutaraldehyde (Sigma, USA). Non-specific binding of antibody wasblocked by using skimmed milk (3.0% in PBS). Cells were incubatedovernight at 4° C. with anti-ICAM-1 mAb (BD, USA), diluted in blockingbuffer, the cells were further washed with PBS and incubated withperoxidase-conjugated goat anti-mouse secondary antibody (Sigma, USA).After washings, cells were exposed to the peroxidase substrate(o-phenylenediamine dihydrochloride 40 mg/100 ml in citrate phosphatebuffer, pH 4.5). Reaction was stopped by the addition of 2 N sulfuricacid and absorbance at 490 nm was measured using microplate reader(Spectramax 190, Molecular Devices, USA).

Example 19 Neutrophil Isolation

Neutrophils were isolated from peripheral blood of healthy individuals(Kumar S et al, 2005). Blood was collected in heparin solution (20 U/ml)and erythrocytes were removed by sedimentation against 6% dextransolution. Plasma, rich in white blood cells, was layered overFicoll-Hypaque solution (Sigma, USA), followed by centrifugation (300 gfor 20 min, 20° C.). The top saline layer and the Ficoll-Hypaque layerwere aspirated leaving neutrophils/RBC pellet. The residual red bloodcells were removed by hypotonic lysis. Isolated cells were washed withPBS and resuspended in PBS containing 5 mM glucose, 1 mM CaCl₂, and 1 mMMgCl₂ at a final concentration of 6×10⁵ cells/ml.

Example 20 Cell Adhesion Assay

Neutrophil adhesion assay was performed under static conditions asdescribed previously (Kumar S et al, 2005). Briefly, endothelial cellsplated in 96-well culture plates were incubated with or without RS—Z atdesired concentrations for 2 hrs, followed by induction with LPS (1μg/ml) for 6 hrs. Endothelial monolayers were washed with PBS andneutrophils (6×10⁴/well) were added over it and were allowed to adherefor 1 hr at 37° C. The non-adherent neutrophils were washed with PBS andneutrophils bound to endothelial cells were assayed by adding asubstrate solution consisting of o-phenylenediamine dihydrochloride (40mg/100 ml in citrate phosphate buffer, pH 4.5), 0.1% cetrimethylammonium bromide, and 3-amino-1,2,4 triazole (1 mM). The absorbance wasread at 490 nm using an automated microplate reader (Model 680, Bio-Rad,USA).

Example 21 Animal Challenge

BALB/c male mice 10 weeks old were randomly divided into 5 groups with 6mice in each group.

Group 1: saline challenged/vehicle (0.25% CMC) treated

Group 2: LPS challenged/vehicle treated

Group 3: LPS challenged/RS—Z (0.1 mg/kg in 0.25% CMC) treated

Group 4: LPS challenged/RS—Z (1 mg/kg in 0.25% CMC) treated

Group 5: LPS challenged/RS—Z (10 mg/kg in 0.25% CMC) treated

Mice in Groups 2, 3, 4 and 5 were challenged with an aerosol of LPS (E.coli strain 026:B6) at a concentration of 300 μg/ml in normal saline for30 minutes. Mice were placed in a Plexiglas chamber (20×20×10 cm³) andexposed to an aerosol generated from a nebulizer (Omtron model, USA)with an airflow rate of 9 L/min. Group 1(Control) mice were challengedwith saline alone.

Example 22 Treatment of RS—Z

Mice were treated with either vehicle or RS—Z thirty minutes after theLPS challenge. Mice in group 1 (control) and Group 2 (LPS challenged)were vehicle (0.25% CMC) treated intraperitoneally (i.p.). Mice in Group3 were treated with RS—Z at a dose of 0.1 mg/kg body weight by i.p.injection. Mice in Group 4 were treated with RS—Z at a dose of 1.0 mg/kgbody weight by i.p. injection. Group 5 were treated with RS—Z at a doseof 10 mg/kg body weight by i.p. injection.

Example 23 RS—Z Reduces Neutrophils in Bronchoalveolar Lavage (BAL)Fluid

Mice were sacrificed three hrs after the LPS challenge using an overdoseof sodium pentothal (100 mg/kg, i.p.). The trachea were cannulated and0.5 ml of PBS (phosphate buffered saline) was used for lavage at a timeand this step was repeated three times. About 1.5 ml of BAL fluid wasrecovered per mouse. The BAL fluid was centrifuged (400×g, 4° C., 6 min)and the supernatant was kept at −70° C. until analyzed for biochemicalparameters. The BAL cells were washed three times with PBS and thepellet was resuspended in 200 μl cold PBS. For total cell counts, BALcell suspension was diluted 1:20 in PBS and cells were counted in ahemocytometer. For differential counts, BALF cell suspension smear wasmade on glass slides followed by Leishman's stain. The cells wereidentified and counted by standard methodology. At least 300 cell (alltypes) per slide were counted and the percentage of neutrophils wascalculated.

Example 24 RS—Z Reduces the Lung Injury Score in Mice

The excised lung portions from all the groups was fixed in 10% Bufferedformalin. The fixed tissue was embedded in paraffin, sectioned into 4 μmand stained with haematoxylin-eosin for observation under the lightmicroscope. The injury was scored with a semi-quantitative gradingsystem based on the structure changes edema, alveolar and interstitialhemorrhage and inflammatory cells sequestration (Matthay M A, ZimmermanG A, Esmon C, Bhattacharya J, Caller B, et al. (2003) Future researchdirections in acute lung injury: Summary of a National Heart, Lung, andBlood Institute working group. Am J Respir Crit. Care Med 167:1027-1035). Semi-quantitative grading system was a 0-to-4 point gradingsystem: 0=No injury, 1=25% injury in a light microscope field, 2=50%injury in a light microscope field, 3=75% injury in a light microscopefield, 4= almost 100% injury in a light microscope field. Five lightmicroscopic fields were analyzed for a particular pathological specimento determine mice lung injury score.

Advantages of the Invention

-   1. The present invention provides novel anti-inflammatory lead    compounds.-   2. This novel molecule can be used to develop new drugs for treating    various inflammatory diseases of humans.-   3. The compounds of present invention are useful as an active    ingredient of anti-inflammatory medicament of acute lung injury or    related conditions like ARDS.

We claim:
 1. A compound of general formula 1

wherein R₁=H, R₂═COOMe, R₃═(CH₂)_(n)COOR₄, R₄═C₂H₅, C₃H₇, C₄H₉, C₅H₁₁,or C₆H₁₁, and n=1-7; or wherein the compound is selected from the groupconsisting of

3-(2-pyridylthio) propionic acid butyl ester (RS20),

3-(2-pyridylthio) propionic acid pentyl ester (RS Z),

7-(2-pyridylthio) heptanoic acid ethyl ester (RS 32), and

3-(2-pyridylthio) propionic acid cyclohexyl ester (RS 21).
 2. Thecompound as claimed in claim 1, wherein the compound is selected fromthe group consisting of

3-(2-pyridylthio) propionic acid butyl ester (RS20);

3-(2-pyridylthio) propionic acid pentyl ester (RS Z);

7-(2-pyridylthio) heptanoic acid ethyl ester (RS 32);

3-(2-pyridylthio) propionic acid cyclohexyl ester (RS 21);

2-(2-pyridylthio)-hexanedioic acid 6-cyclohexyl ester 1-methyl ester (AD21);

2-(2-pyridylthio)-hexanedioic acid 1-methyl ester 6-propyl ester (AD20);

2-(2-pyridylthio)-hexanedioic acid-1-methyl ester 6-pentyl ester (AD Z);and

2-(2-pyridylthio)-decanedioic acid-1O-ethyl ester 1-methyl ester (AD32).
 3. The compound as claimed in claim 1, wherein said compound isuseful as anti-inflammatory agent.
 4. A process for the preparation ofthe compound of claim 1, wherein said process comprising the steps of:i. providing a barton ester of formula IV;

wherein R is —CH₂CH₂COOC₄H₉, —CH₂CH₂COOC₅H₁₁, —(CH₂)₆COOEt,—CH₂CH₂COOC₆H₁₁, or —C(R₁)(R₂)(R₃), wherein R₁═H, R₂═COOMe,R₃═(CH₂)_(n)COOR₄, R₄═C₂H₅, C₃H₅, C₃H₇, C₄H₉, C₅H₁₁, or C₆H₁₁, andn=1-7; ii. diluting the barton ester as provided in step (i) with asolvent up to 25 ml; iii. irradiating the diluted barton ester under sunlight at temperature in the range of 15 to 30° C. for period in therange of 15 to 20 minutes; iv. optionally irradiating the diluted bartonester with olefin under sun light at temperature in the range of 25 to30° C. for period in the range of 15 to 20 minutes; v. removing thesolvent from the irradiated solution as obtained in step (iv) underreduced pressure in the range of 30 to 50 millibar to obtain an oilycrude product; and vi. purifying crude product as obtained in step (v)by preparative TLC to obtain the compound of claim
 1. 5. The process asclaimed in claim 4, wherein solvent used in step (ii) is dry or degassedbenzene or CH₂CI₂.
 6. The process as claimed in claim 4, wherein olefinused in step (iv) is methyl acrylate.
 7. The process as claimed in claim1, wherein step (iii) uses an electric bulb of 200 watt to irradiate thesolution for period in the range of 8 to 10 hrs.
 8. The compound asclaimed in claim 1, wherein said compounds exhibiting inhibition of theLPS induced ICAM-1 (Intercellular cell adhesion molecule-1) expressionand neutrophil adhesion on human endothelial cells with IC₅₀ in therange of 50+0.84 to 178±0.81 μM and 61±0.84 to 94±0.92 μM respectively.9. The compound as claimed in claim 2, wherein compound RS—Z exhibitingreduction of the neutrophil influx in the lungs in a mice model of acutelung injury at doses of 0.1, 1.0 and 10 mg/kg b. wt. i.p. andattenuation of the LPS-induced lung injury in mice at doses of 0.1. 1.0and 10 mg/kg body weight, i.p.